Morphological and functional development of the ... - Science Direct

Brain Research, 580 (1992) 288-296 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

288 BRES 17736

Morphological and functional development of the suprachiasmatic nucleus in transplanted fetal hypothalamus Rafil Aguilar-Roblero a, Shigenobu Shibata b, Joan C. Speh c, Ren6 Drucker-Colfn a and R o b e r t Y. M o o r e c "Departamento de Neurociencias, lnstituto de Fisiolagia Celular, Universidad Nacional AutOnoma de Mdxico, Mdxico, DF (Mdxico), bDepartement of Pharmacology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan) and CDepartments of Psychiatry, Neurology and Behavioral Neuroscience and the Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15261 (USA) (Accepted 31 December 1991)

Key words: Suprachiasmatic nucleus; Neural transplant; Circadian rhythm; Neuronal firing rate; Glucose utilization

The development of the suprachiasmatic nucleus (SCN) in fetal rat hypothalamus transplanted to the adult brain was studied using morphological and functional methods. Anterior hypothalamic tissue was transplanted into the third ventricle, lateral ventricle or subarachnoid space of intact, adult hosts from El7 fetuses. These transplants developed the cytoarchitectonic and immunohistochemical staining characteristics of SCN, clusters of parvocellular neurons expressing vasopressin- and vasoactive intestinal polypeptide-like immunoreactivity in adjacent cellular populations, irrespective of the exact location of the transplanted tissue in the host brain. The functional status of the translants placed in the rostral third ventricle and the foramen of Monroe was analyzed and compared to host SCN using in vitro recording of neuronal firing rate and measurement of metabolism using the 2-deoxyglucose (2-DG) technique. During subjective day, neuronal firing rates and 2-DG uptake were high in discrete cell groups within the transplants which were subsequently demonstrated to exhibit the cytoarchitectonic and immunohistochemical characteristics of SCN. The firing rates and 2-DG uptake in these areas were lower during the subjective night. This pattern of activity closely resembles that of the intact SCN. In contrast, neither transplanted anterior hypothalamic area, lacking an identifiable SCN-like structure, nor posterior hypothalamic area showed day-night differences in firing rate or 2-DG uptake. These observations indicate that SCN transplanted into intact adult hosts exhibits morphological and functional differentiation nearly identical to the host and that the transplanted SCN maintains circadian function which is probably entrained to the host SCN. INTRODUCTION Transplantation of fetal neural tissue provides a useful model for the analysis of development and for studying restoration of function after injury. A m o n g the several situations in which transplants are known to restore function is with the loss of circadian function that results from ablation of the suprachiasmatic nucleus (SCN) of the hypothalamus. In this situation, transplantation of fetal anterior hypothalamus to the third ventricle of atrhythmic hosts results in recovery of the circadian rhythm in locomotor activity 1'11"13'37. There are at least two possible interpretations of these observations. O n e is that the transplanted tissue exerts some 'trophic' effect that induces circadian pacemaker function in the host brain. This would imply that the transplant need not itself express circadian function. A second interpretation is that the transplanted tissue contains a pacemaker that drives effector systems in the host brain that exhibit circadian rhythms. This seems the most likely alternative, partic-

ularly in view of a recent study demonstrating that recovered circadian function in hamsters with a mutation affecting flee-running circadian period reflects the period of the transplant donor, not the host 33. It is now clear that hypothalamic transplants containing SCN develop morphological features that are very similar to intact brain 11'23. There also are data indicating that location of the transplant is important in determining its functional status 1~. It has not been established, however, whether the functional effects of transplants are mediated by direct neural connections with the host brain or by some indirect, humoral mechanism. In the present study fetal anterior hypothalamus containing the SCN was transplanted to multiple brain sites in intact, adult rats. The study had three objectives: to determine the extent to which differentiation of the transplants are affected by location in the host brain; to assess directly the functional status of the transplants; and to determine whether there is interaction between the host and transplanted SCNs.

Correspondence: R. Aguilar-Roblero, Departmento de Neurociencias, Instituto de Fisiologfa Celular, Universidad Nacional Aut6noma de M6xico, Apdo. Postal 70-600, M6xico 04510, DF, M6xico.

289 MATERIALS AND METHODS

Animals Adult male Sprague-Dawley rats (Taconic) weighing between 175 and 200 g were used as transplant hosts in this study. Pregnant female rats, timed by the vendor (Taconic) to be at gestational day 17, were used to provide fetal tissue. The animals were housed in a room maintained in a 12:12 light-dark cycle (lights on at 06.00 h); food and water were available at all times.

Surgery All surgical procedures were performed under aseptic conditions. Subjects were anesthetized with metoxifane vapor (Metofane) followed by intramuscular administration of ketamine/xylazine mixture (Vetalar/Rompun 5:1/ml) at a dose of 1 ml/kg of body weight. The pregnant female rats were anesthetized, the uterus was exposed and one fetus was taken at a time for transplantation. Brains were removed rapidly and placed in sterile saline solution, the anterior hypothalamus was obtained under a dissecting microscope and placed stereotactically into the brain of an anesthetized host through a stainless steel cannula (20 gauge). The procedure from incision into the uterus to the injection of the tissue into the brain was accomplished in less than 3 min, no more than 6 fetuses were taken from each pregnant rat. The level of anesthesia and cardiorespiratory stability of the mother were monitored throughout the experiment in order to ensure the viability of the fetuses remaining in utero. Both the uterine and abdominal wounds were closed after removal of each fetus. At the end of the session, the donor female rat was euthanized with an overdose of anesthesia.

Expt. I: morphological differentiation of transplants Sixteen animals divided into four groups were used in this experiment. The placement of the grafts was as follows; four were placed in the rostral third ventricle, four in the caudal third ventricle, four in the lateral ventricle and four in a cavity in the occipital cortex overlying the superior colliculus. The cavity was prepared by aspiration of the occipital cortex and caudal hippocampal formation. After placement in the cavity, the transplant was secured in place by a piece of gelfoam.

Tissue processing After a survival of 4-6 weeks, the animals received a lethal dose of anesthesia and were perfused transcardially with 50 ml of 0.9% saline solution with heparin (1000 IU/1), followed by 300 ml of fixative (4% paraformaldehyde, 15% saturated picric acid, 0.08% glutaraldehyde in 0.1 M phosphate buffer pH 7.2). The brains were removed and postfixed overnight at 4°C in fixative without glutaraldehyde, and transferred to 20% and 30% sucrose solutions in 0.1 M phosphate buffer (PB), pH 7.2, until they sank. All brains were frozen and sectioned on a freezing microtome in the coronal plane at 30 #m through the rostrocaudal extent of the hypothalamus or the cortical cavity. Sections were collected in ice-cold 0.1 M PB at 150-ktm intervals and divided into four sets for Nissl stain and immunohistochemistry for vasopressin (VP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY).

Immunohistochemical procedures The tissue was processed for immunohistochemistry according to the advidin-biotin peroxidase complex (ABC) method of Hsu 19. Primary antibodies were diluted in 0.1 M PB containing 1.0% normal goat serum in 0.3% Triton X-100 as follows: anti-VP (immunonuclear) 1:2000; anti-VIP (Immunonuclear) 1:2000; and antiNPY (Peninsula labs) 1:2000. The sections were incubated in the primary antibody for 72 h at 4°C, rinsed in PB and incubated for 2 h at room temperature in biotinylated goat anti-rabbit IgG, rinsed in PB and incubated for 2 h at room temperature in avidin-biotin peroxidase complex, rinsed in PB, preincubated 5 rain in 0.05% diaminobenzidine in Tris buffer, pH 7.2, and then reacted for 5 min by adding 35 /~1 of 30% hydrogen peroxide. The reaction was

stopped by rinses in PB. The sections were mounted on gelatin coated slides from a mounting solution (0.1% gelatin in 80% ethanol in PB) and then air dried overnight. The reaction product was intensified by dipping the sections in 0.1% osmium tetroxide in 0.1 M PB for 15-30 s. The sections were then dehydrated in ethanol, cleared with xylene and coverslipped with Permount. The specificity of the antibodies was previously assessed by preabsorbing each antiserum with a 10 ~m solution of the appropriate antigen (VP, Sigma; VIP, Sigma; NPY, Bachem) which was sufficient in each case to block all immunoreactivity. Neurons and axons exhibiting immunoreactivity to the antisera noted above will be designated VP+, VIP+ and NPY+, respectively.

Expt. 2: functional differentiation of transplants Eighteen animals were used for the functional, in vitro experiments. Twelve animals received transplants into the third ventricle from anterior hypothalamic area and six from the posterior hypothalamic area as described in Expt. 1. Intact recipients were used in order to have an internal control for the slice condition and an estimate of the phase of the circadian rhythm from the host. One month after transplantation all the animals were housed in a reversed light-dark cycle (lights on at 19.00 h) for 2 weeks before sacrifice. Two of the SCN grafted animals were housed in constant darkness for 10 days prior to sacrifice and locomotor activity was recorded in these animals in order to establish the phase of an overt rhythm.

Brain slices The rats were decapitated under metoxifane anesthesia and the brain was removed, placed in oxygenated Krebs solution and dissected immediately. Slices (400-500/~m) were obtained on a Vibratome or a tissue chopper in the coronal plane. Those containing the transplanted tissue and the host SCN were preincubated in Krebs solution at 37°C at least 30 min. Slices obtained and used during the light portion of the cycle are referred to as 'subjective day' slices whereas those obtained during the dark are referred to as 'subjective night' slices.

Electrophysiology Neuronal activity was recorded extracellularly through glass electrodes filled with 2 M NaCl (DC resistance, 2-10 Mf]). The composition of solution and recording chamber are described elsewhere42. Single neuron activity was recorded alternatively from the host SCN and the transplant during subjective day (CT 3-7, lights on is designated circadian time (CT) 0), then recorded again from the same areas during subjective night (CT 14-17). In each slice, the transplant was indentified and a drawing made of its location and appearance. The drawing was arbitrarily divided into quadrants and units were recorded throughout each quadrant systematically. When units were indentified with the characteristics of SCN neurons, additional units were analyzed from the same area. The placement of the electrode within the grafted tissue was carefully noted throughout the experiment for later correlation with the results of the immunohistochemistry. The activity recorded from the SCN area was analyzed separately from that obtained from the remaining areas of the transplant. Only single unit activity which remained stable for at least 2 rain was used for data analysis. After termination of electrophysiological experiments, the slices were either fixed in Somogyi fixative 5° or transferred into another chamber for analysis of glucose metabolism. Sections were prepared for immunohistochemical analysis as noted for Expt. 1. In order to establish whether day/night differences existed in the neuronal firing rate, the data were analyzed by the t-test for paired samples, the a level selected was 0.05.

2-Deoxyglucose uptake Glucose metabolism was analyzed using a modification of the 2-DG method developed for use with brain slices3t'32. Incubations with the isotope were carried out for 26 min in 15 ml of solution

290 containing 1 /~Ci/ml of 2-DG (1J4C-2-deoxy-D-glucose, spec. act. 50 mCi/mol, Sigma). The slices were then removed from the chamber and rinsed for 30 rain. Slices were cryostat sectioned at 20 pro, and dried sections were exposed against Kodak OM1 film for two weeks. After exposure, sections were stained with Cresyl violet for histological analysis. The autoradiographs were then analyzed by densitometry and an estimate of local glucose utilization (ELGU, pmol glucose/t00 g tissue/min) was obtained as discribed elswere 31" 32. Differences in the day/night glucose consumption were determined by an ANOVA followed by a Student's t-test for independent samples, the a level selected was 0.05.

RESULTS

Expt. i: morphological differentiation of transplants All of the transplants survived regardless of their placement in the host brain and were located as follows; three in the rostral aspect of the third ventricle (from the lamina terminalis to the rostral half of the paraventricular nuclei), four in the caudal aspect of the third ventricle (from the caudal half of the paraventricular nuclei to the pineal recess), three in the lateral ventricle, two

in the interventricular foramen and four in the cortical cavity, at the dorsolateral aspect of the superior colliculus. The a p p e a r a n c e of the transplants was nearly identical in all locations both in Nissl stain and after immunohistochemical staining. It is thus conceivable that location in the host brain does not m a r k e d l y affect the differentiation of the transplants. F o r this reason, a single description of the transplants is provided. In Nissl stain, there are a b u n d a n t normal appearing neurons, glia, blood vessels and neuropil in the transplants with some glial reaction and a few macrophages apparent in the zone around the host/graft interface, indicating some degree of response from the host. This is minimal in most instances and the host/graft interface is characterized usually by a continuous neuropil b e t w e e n transplant and host brain or an e p e n d y m a l layer along the host ventricular lining (Figs. 1A and 2A). Neurons within the transplant are oval to r o u n d in shape ranging in size from small (less than 15 g m ) to m e d i u m (15-30 #m) with an occasional large neuron

~:., ; ,?5

Fig. 1. Hypothalamic transplant located in foramen of Monro and extending into lateral ventricle. Coronal sections. A: Nissl stain showing two clusters of tightly packed, small neurons in the transplant. The host-transplant interface is marked by asterisks• B-D: adjacent sections• B: NPY immunohistochemistry showing a cluster of neurons (arrow) lying between the two SCN--like cell groups. C: VP+ neurons and fibers in the SCN-like cell groups. D: VIP+ neurons and fibers in the SCN-like cell groups. FM, foramen of Monro; sfo, subfornical organ; th, thalamus. Bar = 100/~m.

291 (greater than 30/~m). T h e cytoarchitecture in the grafts is complex and there is some variability b e t w e e n the transplants and within the same transplant but there are several c o m m o n patterns. These include clusters of small, tightly p a c k e d neurons, usually along the b o r d e r s of the transplants, with an a p p e a r a n c e like SCN in n o r m a l brain. In o t h e r areas the b o r d e r s a p p e a r to be primarily comprised of neuropil. O t h e r regions within the transplants typically contain scattered small to m e d i u m sized neurons within an extensive neuropil and with an app e a r a n c e similar to n o r m a l anterior h y p o t h a l a m i c area. In m a n y transplants there are clusters of small nuclei which a p p e a r glial and are s u r r o u n d e d by concentric or radial arrays of m e d i u m sized neurons. I m m u n o h i s t o c h e m i c a l analysis reveals a very consistent pattern. In the zones with an SCN-like a p p e a r a n c e in Nissl stain, there are clusters of V P + and V I P + neurons which are adjacent and overlapping at the b o r d e r (Figs. 1 and 2). B o t h V P + and V I P + neurons give rise to axon which branch extensively and provide dense plexuses in the vicinity of the neurons T h e a p p e a r a n c e of V P + and V I P + neurons is very similar to n o r m a l SCN. In some transplants there is m o r e than one set of V P + and V I P + neurons. V I P + axons extend out of the i m m e d i a t e , SCN-like zone and often form an axonal plexus along the b o r d e r of the transplant. Ih situations in which there is a continuous neuropil across the bord e r b e t w e e n the transplant and host, there often are both V P + and V I P + axons crossing the b o r d e r . It is not absolutely clear whether these arise from the host, the

transplant or both but some do a p p e a r to arise from V I P + or V P + neurons in the transplant and extend for a short distance into the host brain. T h e r e is a considerable variation in the a m o u n t of N P Y immunoreactivity among the transplants regardless of its placement. In some cases there are n u m e r o u s N P Y + neurons and fibers throughout the extent of the transplant, whereas in others only scattered fibers are found. N P Y + neurons are m e d i u m sized and frequently a p p e a r bipolar. N P Y + fibers are thick and varicose forming a dense plexus within the transplants. N P Y + fibers, when abundant, form a capsule a r o u n d the VPand VIP-containing neurons. Only in one graft placed in the f o r a m e n of M o n r o e N P Y + fibers project into a cluster of V I P + neurons, since only few N P Y + neurons were found within the graft, it is p r o b a b l e that such fibers could arise from the host brain. Projections of

TABLE I Diurnal changes in electrical and metabolic activity in hypothalamic slices in vitro

aMean -+ S.D. of neuronal firing rates (Hz). The slices were prepared from four animals and then recorded at two different circadian times (CT). bMean + S.D. of the local glucose utilization ~ M glucose/100 g tissue/min). The slices were prepared from eight animals. Four subjects were used at each CT, except for the grafts from posterior hypothalamus (PHA). The SCN and AHA areas in the transplant are designated by their neuronal firing rate characteristics and confirmed by immunohistochemistry. SCN, suprachiasmatic nucleus; AHA, anterior hypothalamic area. ElectricaP

HOST

SCN AHA GRAFT SCN AHA PHA

Metabolic b

CT 4-6

CT 16-18

CT 4-6

7.2_+2.8* 2.9+1.3 8.1+5.8" 2.4+1.6 -

3.4_+2.1 2.6+0.9 2.7-+1.8 3.2-+1.9 -

47.2+6.2** 25.6+1.3 13.0+2.2 12.7+1.2 43.0-+2.6** 22.8+3.2 9.8_+1.3 12.0+2.1 10.8+0.9 9.0+1.3 (n = 3) (n = 3)

i~~ "

CT 16-18

*P < 0.05 (host, t = 2.8; graft, t = 5.1. Paired t-test). **P < 0.05 (host, F = 46.5; t 3 4.8, graft, F = 96, t = 6.9).

l @

Fig. 2. Hypothalamic transplant located in lateral ventricle. This transplant was used for electrophysiological recording and subsequently prepared for immunohistochemistry. Coronal sections. A: Nissl stain; B: VP immunohistochemistry; C: VIP immunohistochemistry. G, transplant; LV, lateral ventricle. Bar -- 100/~m.

292

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iliir

Fig. 3. Coronal sections from hypothalamic slices obtained during subjective day and prepared for analysis of glucose utilization. A-C left: Nissl stains, A: host SCN (lying above optic chiasm, OC); B: transplant from same brain located in third ventricle (asterisk indicates SCNlike cell cluster); C: transplant from a different brain. Right, autoradiographs from sections shown in A-C. There is high glucose utilization in the host SCN and in the SCN-like clusters in the transplants.

N P Y + axons into the host brain appear limited to the vicinity of the transplant b o u n d a r y regardless of its placement.

Expt. 2: electrophysiology Single n e u r o n activity was recorded from both the host SCN and the transplant during subjective day (CT

293 4-6), then recorded again from the same areas during subjective night (CT 16-18). Eight slices from four animals, one containing the host's SCN and one the grafted tissue, were used in this experiment. Once the records were completed, the slices were processed for immunohistochemistry as previously described. The firing rates of neurons within the host SCN is higher than the surrounding hypothalamic areas which are characterized by neurons with slow and irregular firing rates. Host SCN neurons typically show three firing patterns as discribed previously 15'16'40-42. These are a regular firing pattern (type I), an irregular firing pattern (type II) and a bursting firing pattern (type III). Typical day-night differences in the firing rates and firing pattern are found in the host SCN. During subjective day, most SCN neurons discharge tonically with a type I pattern with a constant interspike interval at mean firing rate of 7.2 _+ 2.8 Hz. During subjective night the firing rate decreases to 3.4 _+ 2.1 Hz with many neurons exhibiting a type II pattern. A type III firing pattern is only occasionally observed. The pattern of electrical activity within the transplanted tissue is variable, ranging from extensive areas containing units with irregular and low firing rates to discrete regions with high spontaneous activity. Those areas with the higher levels of activity correspond to an SCN-like structure defined by Nissl stain and immunohistochemistry. Neuronal firing rates and patterns from the SCN-like areas show day-night differences similar to those found in the host SCN. During subjective day most of these neurons exhibit a type I pattern with a mean firing rate of 8.1 + 5.8 Hz. During subjective night, a type II pattern is more frequently found and the neurons have a mean firing rate of 2.7 _+ 1.8. In the other areas of the transplants there are no differences found either in firing rates or pattern between subjective day and night (see Table I). The same patterns of electrical activity are found in slices from animals housed in constant darkness for 10 days prior to sacrifice. The timing of recording was determined from analysis of locomotor activity obtained for the 3 days prior to sacrifice. The onset of locomotor activity was designated as CT 12. The electrical activity in the host SCN and the SCN-like areas within the graft show day-night differences in firing rate and pattern similar to those observed in slices from those animals housed in light-dark cycles.

Expt. 2: 2-DG uptake Single neuron activity was recorded from both the host SCN and the transplanted tissue, and the slice was then transferred from the recording chamber to another one in which the slice was incubated with [laC]2-DG. It

was then processed for autoradiography. These procedures were done in two groups of slices at different CTs, one during subjective day (CT 6) and the other during subjective night (CT 18). Ten to fourteen slices from seven animals were used in each group for this experiment, one or two slices from each animal were used depending on the placement of the graft. At each circadian time four animals grafted with anterior hypothalamus and three with posterior hypothalamus were used. Nissl staining of the sections showed clusters of tightly packed, parvocellular neurons similar to those found in the normal, adult SCN. In three animals with posterior hypothalamic grafts, no SCN-like structure was found. Electrical activity recorded both from the host SCN and the SCN-like areas within the transplant shows d a y night differences in firing rate and pattern similar to those described in the previous experiment. That is, the electrical activity in the host SCN and in the SCN-like areas in the transplant are higher during subjective day than during subjective night (these data are not shown and were used only to locate the SCN-like area within the graft). The remaining areas of the transplant and host anterior hypothalamic area (AHA) have irregular firing patterns and low firing rates with no circadian variation. No day-night differences in either firing rate or pattern are found among neurons recorded from the posterior hypothalamic transplants. Autoradiograms from the slices containing both the host SCN and the SCN-like areas incubated during subjective day are shown in Fig. 3. The autoradiograms were used to generate data for E L G U . Since the mean values for right and left host SCN and A H A were comparable for each rat, these values were averaged 31'32. During subjective day, the mean E L G U in the host SCN is 47.2 + 6.2, higher than the surrounding hypothalamic areas (13.0 + 2.2), see Table I. In the transplanted tissue a similar pattern was found; the E L G U is 43.0 __+ 2.6 in the SCN-like areas and 9.8 + 1.3 in the surrounding tissue. During subjective night the mean E L G U in the host SCN is 25.6 + 1.3, lower than found during subjective day. In contrast the mean E L G U from the host A H A (12.7 + 1.2) during subjective night does not differ from the value observed during subjective day in the same area. A similar pattern is found in the transplanted tissue, E L G U is 22.8 + 3.2 in the SCN-like areas and 12.0 + 2.1 in the surrounding regions. In those animals with transplanted posterior hypothalamic tissue, there is a significant day-night difference in the E L G U in the host SCN but, as would be expected, not in the transplanted tissue. For transplants containing SCN, the day-night differences in E L G U in both the host SCN and the transplanted SCN are similar so that the day/ night ratio is exactly the same (1.9) for both cases. These

294 data are similar to those reported

previously 31'32'39'42.

DISCUSSION The results of the present study show that fetal hypothalamic tissue routinely survives and differentiates when transplanted to different areas of the central nervous system of adult hosts. In addition to morphological differentiation, the transplanted tissue exhibits circadian function with rhythms in electrical and metabolic activity in the regions of the transplant that have an SCN-like appearance. The SCN is readily identified in Nissl material in adult brain as a pair of nuclei containing compact groups of small neurons lying dorsal to the optic chiasm and adjacent to the third ventricle. In the rat, the SCN has two subdivisions which are most readily indentified by immunohistochemistry 21'27'46. The dorsomedial division is characterized by a large population of VP- and neurophysin-containing neurons 43'46 and smaller populations of somatostatin 12'18 and enkephalin-containing 27 neurons. The dorsomedial SCN does not receive retinal afferents 2~. In contrast, the ventrolateral SCN receives a dense retinal projection 21, a dense projection of NPYcontaining axons from the intergeniculate leaflet 8'3° and is characterized by a large population of VIP-containing neurons 9 and smaller populations of gastrin releasing peptide- and neurotensin-containing neurons 46. This information concerning chemically characterized neurons in SCN subdivisions has made it possible to identify SCN in fetal anterior hypothalamic tissue transplanted to the brain 23'49 and anterior chamber of the eye 34. In both circumstances, the differentiation of SCN subdivisions is very similar to that in the normal brain. The present study confirms these observations. The transplanted tissue in our material has areas of densely packed parvocellular neurons which, on immunohistochemical staining, contain partially overlapping groups of VP- and VIP-containing neurons with extensive axonal arborizations extending over much of the parvocellular neuron area. These observations indicate that, like other areas of fetal brain 2'17'22'24-26 fetal SCN is programmed such that it is able to differentiate in an environment quite different from its normal location. The amount of NPY immunoreactivity varied among transplants from numerous cells and fibers to only scattered fibers. In all cases in which N P Y + neurons were abundant in the transplanted tissue, they were outside the SCN-like area and produced extensive plexuses of fibers which usually formed a capsule surrounding the SCN-like area. A similar picture, that is a dense NPY fiber plexus surrounding the SCN without actually entering the nuclei, has been seen after lesions of the inter-

geniculate leaflet (Johnson, Morin and Moore, personal communication). Hence, it is suggested that hypothalamic NPY neurons do not duplicate the pattern of NPY innervation of SCN normally provided by the intergeniculate leaflet. In that case in which N P Y + fibers were found invading the grafted SCN, only a few N P Y + neurons were found through the graft extent. Thus, it is possible that such fibers arise from the host brain rather than from the NPY neurons from the graft. In all of the transplants with continuous neuropil extending from the transplant to host brain, V P + , V I P + and N P Y + fibers are observed crossing the interface of transplant and host neuropil, regardless of the placement of the transplanted tissue. For the most part, the fibers appear to originate from the transplant and extend no more than a short distance into the host brain. These fibers form a loose plexus with no apparent pattern of connectivity. This is in contrast to some situations where very specific patterns of connectivity are established between transplant and host. For example, catecholamine neurons innervating hippocampal formation form patterns similar to those in intact brain 3-5, fetal dopamine neurons in suspension innervate the neostriatum but not the substantia nigra or lateral hypothalamus 6'7, and fetal entorhinal cortex specifically reinnervates hippocampus and amygdala denervated by transection of the angular bundle 14. In mice, the pattern of fiber growth from fetal hippocampal tissue in reinnervating host hippocampus follows the pattern established in normal development 51. To analyze the functional status of the transplant, single unit activity was recorded systematically from host SCN and adjacent anterior hypothalamic area and throughout the transplant. Discrete regions of the transplants contain neurons with high firing rates and regular firing patterns indentical to neurons in the host SCN during subjective day. When the same region was recorded during subjective night, the mean firing rate was substantially reduced and a greater proportion of neurons exhibited irregular firing patterns. This change in firing pattern is also found in the host SCN and both aspects of the rhythm are typical of intact SEN 20'40-42. The areas of the transplant which exhibit these electrophysiological features also show rates of glucose utilization that are similar to the host SCN and contain populations of parvocellular neurons which stain for either VP or VIP. In contrast, adjacent portions of transplant have the electrophysiological, metabolic and morphological characteristics of anterior hypothalamic area. Posterior hypothalamic transplants also do not show the functional characteristics of SCN nor do they contain any neuronal groups with morpological features similar to SCN. In all instances, the host and transplanted tissue exhibit functional features which appear to be in phase.

295 That is, when firing rate or glucose utilization was high in the host SCN, it was high in the SCN-like area of the transplant. We lack sufficient d a t a to definitively establish that there is an exact c o r r e s p o n d e n c e in phase between host and transplant but the similarity b e t w e e n t h e m in each p r e p a r a t i o n strongly suggests they are in phase. Since the host SCN is innervated by retinal afferents, and entrained by l i g h t - d a r k cycle, it seems likely that the host SCN entrains the SCN-like populations in the transplant. Since this occurred in every case in this study, it implies that the phase and p e r i o d of an entrained p a c e m a k e r will take p r e c e d e n c e over that of a free-running one. The e n t r a i n m e n t of transplanted SCN by host SCN could be accomplished by either neuronal connections or a h u m o r a l effect. T h e r e a p p e a r to be relatively few connections b e t w e e n host and transplant, at least ones d e m o n s t r a b l e by the m e t h o d s e m p l o y e d . M o r e o v e r , the location of the transplants with respect to the host SCN is quite variable. Thus, it would seem

m o r e p r o b a b l e that the communication b e t w e e n host and transplant is humoral. The means by which transplants generate rhythmicity in SCN-lesion hosts is unknown but certainly could be through release of a humoral agent. In our situation, communication of an entraining signal between host and SCN and transplanted SCN, two types of h u m o r a l signal are possible. O n e would be a direct signal from the host SCN, possibly transmitted via the cerebrospinal fluid. A second would be an indirect signal using a h o r m o n e such as melatonin which is known to have entraining effects on the SCN 1°' 47. F u r t h e r experiments are n e e d e d in o r d e r to settle these questions.

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Acknowledgements. Supported by NIH Grant NS-16304. We are grateful to Ms. Priscila Wu for skilled technical assistance and to Mrs. Mary Ann Scriven and Mrs. Teresa Torres for preparing the manuscript.

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